N-Acetylglucosaminyltransferases (GlcNAc-transferases) are “branching” enzymes that add an Nacetylglucosamine (GlcNAc) residue to one of the mannoses of the trimannosyl core structure of typical Nlinked glycans. At least six GlcNAc-transferases are known with little or no sequence homology. Besides different protein structures, these GlcNActransferases also have different enzymatic properties and substrate specificity. All are typical type II transmembrane proteins with a cytoplasmic domain, a transmembrane anchor and an extracellular stem region with catalytic domain.
A remarkable GlcNAc-transferase is GlcNAc-transferase III (GnTIII). GnTIII, also known as UDP-Nacetylglucosamine:β-D-mannoside β(1,4)-N-acetylglucosaminyl-transferase III (EC 2.4.1.144), inserts bisecting GlcNAc residues in complex-type N-linked glycans of cellular glycoproteins (for a review see Taniguchi, et al., “A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes” Proteomics 1:239247, 2001). GnTIII adds the GlcNAc through a β(1,4) linkage to the β-linked mannose of the trimannosyl core structure of the N-linked glycan. GnTIII was first identified in hen oviduct (Narasimhan S., “Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide β 4-Nacetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in β14 linkage to the β-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides” The Journal of Biological Chemistry 257:10235-10242, 1982) but a high level of activity has also been reported in various types of rat hepatomas, human serum, liver and hepatoma tissues of patients with hepatomas and liver cirrhosis (Ishibashi, et al., “N-acetylglucosaminyltransferase III in human serum and liver and hepatoma tissues: increased activity in liver cirrhos and hepatoma patients” Clinical Chimica Acta 185:325, 1989; Narishimhan, et al., “Expression of N-acetylglucosaminyltransferase III in hepatic nodules during rat liver carcinogenesis promoted by orotic acid” Journal of Biological Chemistry 263:1273-1281, 1988; Nishikawa, et al. “Determination of N-acetylglucosaminyltransferases III, IV and V in normal and hepatoma tissues of rats” Biochimica et Biophysica Acta 1035:313-318, 1990; Pascale, et al., “Expression of N-acetylglucosaminyltransferase III in hepatic nodules generated by different models of rat liver carcinogenesis” Carcinogenesis 10:961964, 1989). Bisected oligosaccharides on glycoproteins have been implicated in antibody-dependent cellular cytotoxicity (ADCC). ADCC is a lytic attack on antibody-targeted cells and is triggered upon binding of lymphocyte receptors to the constant region (Fc) of antibodies. Controlled expression of GnTIII in recombinant Chinese Hamster Ovary (CHO) production cell lines that lack GnTIII activity resulted in antibodies with bisected oligosaccharides with optimized ADCC activity (Davies, et al., “Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FcγRIII” Biotechnology and Bioengineering 74:288-294, 2001; Umana, et al., “Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity” Nature Biotechnology 17:176-180, 1999). The ADCC activity correlated well with the level of Fc region-associated bisected complex oligosaccharides present on the recombinant antibody (Umana, et al., “Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity” Nature Biotechnology 17:176-180, 1999). Bisecting GlcNAc residues resulting from GnTIII activity affect the conformation of the sugar chains in such a way that other glycosyltransferases such as GlcNAc-transferase II and α1,6-fucosyltransferase, but not β(1,4)-galactosyltransferase, can no longer act (Tanigichi, et al., 2001). Overexpression of GnTIII in CHO cells is lethal.
In contrast to typical mammalian production cell lines such as CHO cells, transgenic plants are generally recognized as a safe production system for therapeutic proteins. Plant glycoproteins, however, differ in oligosaccharide structure with those from mammals in several aspects. They lack terminal galactose and sialic acid, have an additional core xylose and differently linked core fucose (α-1,3) instead of (α-1,6). Like CHO and other pharmaceutical production cell lines they also completely lack bisected oligosaccharides. Plants have the capacity to generate the common core structure, GN2M3GN2 but predominantly M3 GN2 variants are found, indicating removal of terminal GN by hexosaminidases.
Biogenesis of N-linked glycans begins with the synthesis of a lipid linked oligosaccharide moiety (Glc3Man9GlcNAc2-) which is transferred en bloc to the nascent polypeptide chain in the endoplasmic reticulum (ER). Through a series of trimming reactions by exoglycosidases in the ER and cis-Golgi compartments the so-called “high mannose” (Man9GlcNAc2 to Man5GlcNAc2) glycans are formed. Subsequently, the formation of complex type glycans starts with the transfer of the first GlcNAc onto Man5GlCNAC2 by GnTI and further trimming by mannosidase II (Mann) to form GlcNAcMan3GlcNAc2. Complex glycan biosynthesis continues while the glycoprotein is progressing through the secretory pathway with the transfer in the Golgi apparatus of the second GlcNAc residue by GnTII as well as other monosaccharide residues onto the GlcNAcMan3GlcNAc2 under the action of several other glycosyl transferases. Plants and mammals differ with respect to the formation of complex glycans. In plants, complex glycans are characterized by the presence of β(1,2)-xylose residues linked to the Man-3 and/or an α(1,3)-fucose residue linked to GlcNAc1, instead of an α(1,6)-fucose residue linked to the GlcNAc-1 (Lerouge, P., et al., “N-glycoprotein biosynthesis in plants: recent developments and future trends” Plant Mol Biol 38:31-48, 1998). Genes encoding the corresponding xylosyl (XylT) and fucosyl (FucT) transferases have been isolated (Strasser R, “Molecular cloning and functional expression of β1,2-xylosyltransferase cDNA from Arabidopsis thaliana” FEBS Lett. 472:105-8, 2000; Leiter, H, et al, “Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase from mung beans” J Biol Chem. 274:21830, 1999). Xylose and fucose epitopes are known to be highly immunogenic and possibly allergenic which may pose a problem when plant are used for the production of therapeutic glycoproteins. Moreover, blood serum of many allergy patients contains IgE directed against these epitopes which make particularly these patients at risk to treatments with xylose and fucose containing recombinant proteins. In addition, this carbohydrate directed IgE in sera might cause false positive reaction in in vitro tests using plant extracts since there is evidence that these carbohydrate specific IgE's are not relevant for the allergenic reaction. Plants do not possess β(1,4)galactosyltransferases nor α(2,6)sialyltransferases and consequently plant glycans lack the β(1,4)galactose and terminal α(2,6)NeuAc residues often found on mammalian glycans (Vitale and Chrispeels, “Transient N-acetylglucosamine in the biosynthesis of phytohaemagglutinin: attachment in the Golgi apparatus and removal in protein bodies” J Cell Biol 99:133-140, 1984; Lerouge, P., et al., “N-glycoprotein biosynthesis in plants: recent developments and future trends” Plant Mol Biol 38:31-48, 1998).
The final glycan structures are not only determined by the mere presence of enzymes involved in their biosynthesis but to a large extend by the specific sequence of the various enzymatic reactions. The latter is controlled by discrete sequestering and relative position of these enzymes throughout the ER and Golgi, which is mediated by the interaction of determinants of the transferase and specific characteristics of the sub-Golgi compartment for which the transferase is destined. A number of studies using hybrid, molecules have identified that the transmembrane domains of several glycosyltransferases play a central role in their sub-Golgi sorting (Grabenhorst E., et. al., J. Biol. Chem. 274:36107-36116, 1999; Colley, K., Glycobiology. 7:1-13, 1997, Munro, S., Trends Cell Biol. 8:11-15, 1998; Gleeson P. A., Histochem. Cell Biol. 109:517-532, 1998).
Similar to mammalian production cell lines used in pharmaceutical industry, glycoproteins produced in plants lack GnTIII activity. Plants not only lack GnTIII activity but are completely devoid of GnTIII-like sequences. In addition, plants also lack GnTIV, GnTV ands GnTVI sequences and moreover, sialic acid residues. (For an overview of the major glycosylation attributes of commonly used cell expression systems including plants see, Jenkins, et al., “Getting the glycosylation right: implications for the biotechnology industry” Nature Biotechnology 14:975-979, 1996). Nevertheless, plants are a very potent production system. Plants are generally accepted as safe and are free of particles infectious to humans. Plant production is easy scalable and N-linked glycosylation can be controlled (Bakker, et al., “Galactose-extended glycans of antibodies produced by transgenic plants” Proc. Nat. Acad. Sci. USA 98:2899-2904, 2001).
Transgenic tobacco plants that produce galactosylated recombinant monoclonal antibodies (Mabs) upon introduction of the human gene for β(1,4)-galactosyltransferase have been reported (hGal T; Bakker, et al., “Galactose-extended glycans of antibodies produced by transgenic plants” Proc. Nat. Acad. Sci. USA 98:2899-2904, 2001; WO01/31044 and WO01/31045).
Therapeutic glycoproteins can be improved by altering their glycosylation pattern (Davies, et al., “Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FcγRIII” Biotechnology and Bioengineering 74:288-294, 2001; Umana, et al., “Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity” Nature Biotechnology 17:176-180, 1999; Fukuta, et al., “Remodeling of sugar chain structures of human interferon-γ” Glycobiology 10:421-430, 2000; Misaizu, et al., “Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin” Blood 86:4097-4104, 1995; Sburlati, et al., “Synthesis of bisected glycoforms of recombinant IFN-β by overexpression of β-1,4-N-acetylglucosaminyl-tranferase III in Chinese Hamster Ovary cells” Biotechnology Prog. 14:189-192, 1998). Higher oligosaccharide antennarity of EPO, for example, leads to increased in vivo activity due to reduced kidney filtration (Misaizu, et al., “Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin” Blood 86:4097-4104, 1995). Biosynthesis of such superior glycoforms can be achieved with the “standard” glycosylation machinery of normal production cell lines by two methodologies. The first is by enriching specific glycoforms during purification and the second is by introducing mutations in the polypeptide chain. The latter makes it possible to shift the glycosylation site within the glycoprotein resulting in different glycosylation patterns as the result of differences in accessibility. A complementary route is through genetic engineering of the production cell line itself. New glycosylation patterns can be obtained through expression of glycosyltransferase and glycosidase genes in production cell lines. These genes code for enzymes that either add or remove specific saccharides to and from the glycan of cellular glycoproteins. Several glycosyltransferase genes have been introduced in CHO cells to manipulate glycoform biosynthesis. One of them is GnTIII. Glycosyltransferase GnTIII is involved in branching of the N-linked glycan and results in bisecting GlcNAc residues. CHO cells and other production cell lines typically lack GnTIII activity (Stanley, P. and C. A. Campbell, “A dominant mutation to ricin resistance in chinese hamster ovary cells induces UDP-GlcNAc: glycopeptide β-4-N-acetylglucosaminyl-transferase III activity” Journal of Biological Chemistry 261:13370-13378, 1984). Expression of GnTIII in CHO resulted in bisected complex oligosaccharides as expected but overexpression resulted in growth inhibition and was toxic to cells. Similarly, overexpression of GnTV, another glycosyltransferase that introduces triantennary sugar chains, also resulted in growth inhibition suggesting that this may be a general feature of glycosyltransferase overexpression (Umana, et al., “Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity” Nature Biotechnology 17:176-180, 1999).
Therefore, there is a need to provide a means for producing glycoprotein in plants with human compatible non-immunogenic bisecting oligosaccharides.